We propose to determine the structural requirements of the formylatable and non-formylatable E. coli methionine tRNAs (tRNAfMet and tRNAmMet) for recognition by E. coli methionyl-tRNA synthetase and for attachment of methionine. In addition, we propose to determine the structural requirements for recognition of Met-tRNAfMet by E. coli methionyl-tRNA transformylase and the structural basis for binding of fMet-tRNAfMet to the 30S ribosomal subunit and to the 70S ribosomal P site. The approach to be used involves chemical modification of purified tRNA, separation of the tRNA after modification into components which are active and inactive with respect to the biological activity being examined, and analysis of the structure of the active and inactive molecules for the sites of the modifications. We plan to develop methods for attachment of crosslinking reagents to a variety of sites in tRNAsMet. These tRNAs will then be used to investigate the protein sequences in E. coli methionyl-tRNA synthetase (MRS) which are at or near tRNA binding sites and to probe the topography of ribosomal tRNA binding sites. In addition, we plan to prepare fluorescent-labeled tRNAs for studies of tRNAMet binding to MRS and to ribosomes.